Chlorambucil produces its anti-cancer effects by interfering with DNA replication and damaging the DNA in a cell. The DNA damage induces cell cycle arrest and cellular apoptosis via the accumulation of cytosolic p53 and subsequent activation of Bax, an apoptosis promoter.[7][8][9]

Chlorambucil alkylates and cross-links DNA during all phases of the cell cycle, inducing DNA damage via three different methods of covalent adduct generation with double-helical DNA:[10][11][12]

Attachment of alkyl groups to DNA bases, resulting in the DNA being fragmented by repair enzymes in their attempts to replace the alkylated bases, preventing DNA synthesis and RNA transcription from the affected DNA.

DNA damage via the formation of cross-links which prevents DNA from being separated for synthesis or transcription.

Induction of mispairing of the nucleotides leading to mutations.

The precise mechanisms by which Chlorambucil acts to kill tumor cells are not yet completely understood.

Nitrogen mustards arose from the derivatization of sulphur mustard gas after military personnel exposed to it during World War I were observed to have decreased white blood cell counts.[15] Since the sulphur mustard gas was too toxic to be used in humans, Gilman hypothesized that by reducing the electrophilicity of the agent, which made it highly chemically reactive towards electron-rich groups, then less toxic drugs could be obtained. To this end, he made analogues that were less electrophilic by exchanging the sulphur with a nitrogen, leading to the nitrogen mustards.[16]

With an acceptable therapeutic index in humans, nitrogen mustards were first introduced in the clinic in 1946.[17] Aliphatic mustards were developed first, such as mechlorethamine hydrochloride (mustine hydrochloride) which is still used in the clinic today.

In the 1950s, aromatic mustards like chlorambucil were introduced as less toxic alkylating agents than the aliphatic nitrogen mustards, proving to be less electrophilic and react with DNA more slowly. Additionally, these agent can be administered orally, a significant advantage.